Despite remarkable achievements in the development of vaccines for certain viral infections (i.e., polio and measles), and the eradication of specific viruses from the human population (e.g., smallpox), viral diseases remain as important medical and public health problems. Indeed, viruses are responsible for several “emerging” (or re-emerging) diseases (e.g., West Nile encephalitis and Dengue fever), and viral infection is a cause of significant morbidity and mortality worldwide.
The presence of adequate T-cell help is important for the construction of potent vaccines. Vaccines that induce both helper T cells and CTLs may be more effective that those that induce CTLs only. Indeed, the importance of cooperation between CD4+ and CD8+ T cells is emphasized in the therapeutic vaccination against chronic viral infection (Zajac et al., 1998; Matloubian et al., 1994).
Classically, vaccines are manufactured by introducing killed or attenuated organisms into the host along with suitable adjuvants to initiate the normal immune response to the organisms while, desirably, avoiding the pathogenic effects of the organism in the host. The approach suffers from the well known limitations in that it is rarely possible to avoid the pathogenic response because of the complexity of the vaccine which includes not only the antigenic determinant of interest but many related and unrelated deleterious materials, any number of which may, in some or all individuals, induce an undesirable reaction in the host.
For example, vaccines produced in the classical way may include competing antigens which are detrimental to the desired immune response, antigens which include unrelated immune responses, nucleic acids from the organism or culture, endotoxins and constituents of unknown composition and source. These vaccines, generated from complex materials, inherently have a relatively high probability of inducing competing responses even from the antigen of interest.
HSP60 belongs to a family of chaperone molecules highly conserved throughout evolution; a similar HSP60 molecule is present in all cells, prokaryotes and eukaryotes. The human HSP60 molecule was formerly designated HSP65, but is now designated HSP60 in view of more accurate molecular weight information; by either designation, the protein is the same. Apparently, no cell can exist without the ability to express HSP60. Mammalian HSP60 is highly homologous to the bacterial cognates, showing about 50% amino acid identity (Jindal et al., 1989). Thus, HSP60 is shared by the host and its parasites, and is immunogenic, cross-reactive, and universally expressed in inflammation. Furthermore, HSP60 is well recognized by the immune system (Konen Waisman et al., 1999, Konen Waisman et al., 1995) and is a part of the set of self-molecules for which autoimmunity naturally exists; HSP60 is member of the immunologic homunculus (Cohen, 1992). Heat shock, IFNγ, bacterial or viral infection, and inflammation, all result in the presentation of endogenous HSP60 epitopes on MHC class II molecules leading to the activation of HSP60-specific T cells, even in healthy individuals (Anderton et al., 1993; Hermann et al., 1991; Koga et al., 1989).
European Patent EP 262 710 and U.S. Pat. No. 5,154,923 describe peptides having an amino acid sequence corresponding to positions 171-240 and 172-192, respectively, of a Mycobacterium boris BCG 64 kD polypeptide, that are useful as immunogens inducing resistance to autoimmune arthritis and similar autoimmune diseases.
PCT Patent Application No. WO 90/10449 describes a peptide designated p277 having an amino acid sequence corresponding to positions 437-460 of the human HSP65 molecule that is useful as immunogen inducing resistance to insulin dependent diabetes mellitus (IDDM). A control peptide, designated p278, corresponding to positions 458-474 of human HSP65, did not induce resistance to IDDM.
Lussow et al. (1990) showed that the priming of mice with live Mycobacterium tuberculosis var. Bovis (BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies. Later on, Lussow et al. (1991) showed that mycobacterial heat-shock proteins (HSP) of 65 kDa (GroEL-type) and 70 kDa (DnaK-type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. boris (bacillus Calmette-Guerin, BCG), for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial HSP, was given in the absence of any adjuvants.
Barrios et al. (1992) have shown that mice immunized with peptides or oligosaccharides conjugated to the 70 kDa HSP produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year. This adjuvant-free carrier effect of the 70 kDa HSP was T cell dependent, since no anti-peptide nor anti-70 kDa IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65 kDa or 70 kDa HSP did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with HSP-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65 kDa HSP could substitute for BCG in providing effective priming for the induction of anti-(NANP)40 antibodies. Finally, both the 65 kDa and 70 kDa HSP acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants, suggesting that the use of HSPs as carriers in conjugated constructs for the induction of anti-peptide and anti-oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.
U.S. Pat. No. 5,736,146 discloses conjugates of poorly immunogenic antigens with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of human heat shock protein HSP65, or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the poorly immunogenic antigen. The '146 patent discloses conjugates of a peptide corresponding to positions 458-474 and 437-453 of human or mouse HSP60 and homologs thereof with a wide variety of antigens including peptides, proteins and polysaccharides such as bacterial polysaccharide (e.g. capsular polysaccharide (CPS) Vi of Salmonella typhi), and antigens derived from HIV virus or from malaria antigen.
U.S. Pat. No. 5,869,058 discloses conjugates of poorly immunogenic antigens, e.g., peptides, proteins and polysaccharides, with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of E. coli HSP65 (GroEL), or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the poorly immunogenic antigen. A suitable peptide according to the invention is Pep278e, which corresponds to positions 437-453 of the E. coli HSP65 molecule.
Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus from the betaherpesvirus group; it is endemic in all human populations. In North America, HCMV infects about 50% of the population outside of urban centers and up to 90% of the population within cities. HCMV disease presents two major medical problems: first, it is the most common congenital viral infection, causing birth defects including mal-development of the central nervous system; up to 25% of asymptomatic infected infants will develop neurologic sequelae. Second, HCMV becomes re-activated in immunocompromised patients.
A self-limiting acute phase of viral infection, persistent and latent phases normally characterize the pathogenesis of HCMV infection in the immunocompetent host. The clinical outcome of HCMV infection is determined by the ability of infected individuals to mount protective humoral and T-cell mediated immune responses. In immunocompromised hosts, including persons with HIV infection, cancer patients and allograft recipients, primary HCMV infection or reactivation of a latent virus results in multi-organ HCMV disease, associated with high rates of morbidity and mortality. These grave clinical consequences emphasize the need for effective HCMV vaccines to prevent not only primary infection but also to limit or prevent reactivation.
At present there is no protective vaccine available for CMV. Currently available antiviral drugs which target viral DNA replication are efficacious but exhibit significant host toxicity and a high spontaneous resistance rate.
West Nile virus is a member of the alpha-like Flaviviridae. The Flavivirus genome is a single-stranded, positive-sense RNA approximately 11 kb in length, containing a 5′ untranslated region (5′UTR); a coding region encoding the three viral structural proteins; seven nonstructural proteins, designated NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5; and a 3′ untranslated region (3′UTR). The viral structural proteins include the capsid (C), premembrane/membrane (prM) and envelope (E) proteins. The structural and nonstructural proteins are translated as a single polyprotein. The polyprotein is then processed by cellular and viral proteases.
West Nile virus affects birds as well as reptiles and mammals, together with man. The West Nile virus is transmitted to birds and mammals by the bites of certain mosquitoes (e.g. Culex, Aedes, Anopheles). Direct transmission may happen from WNV infected subject to healthy subject by oral transmission (prey and transmission through colostrum) and blood/organ vectored transmission. Widespread in Africa, the geographic range of WNV now also includes Australia, Europe, the Middle East, West Asia and the USA. West Nile virus can cause a harsh, self-limiting fever, body aches, brain swelling, coma, paralysis, and death.
There is no effective treatment for the disease. A number of different WNV vaccines are now in various stages of development and testing (Monath, 2001; Pletnev et al., 2003; Tesh et al., 2002; Hall et al., 2003), but presently a licensed human vaccine is not available for its prevention. The only currently effective way to provide immediate resistance to WNV is by passive administration of protective antibodies (Casadevall, 2002). Mosquito control is currently considered the practical strategy to combat the spread of disease, but effective spraying is difficult to perform in urban areas. Clearly, an effective vaccine is needed to protect at-risk populations.
There remains a need for improved vaccines conferring protection against viral infections, using isolated epitopes. Furthermore, isolated epitopes are needed for improved diagnostic tests.